1. Field of the Invention
The invention is in the field of recombinant DNA technology and relates to promoters useful for the expression of foreign DNA inserted into a fowlpox virus vector.
2. Description of the Prior Art
Poxviruses are large viruses with a complex morphology containing linear double-stranded DNA genomes. They are among the few groups of DNA viruses that replicate within the cytoplasm of the cell. They are subclassified into six genera: orthopoxviruses, avipoxviruses, capriopoxviruses, leporipoxviruses, parapoxviruses and entomopoxviruses. Vaccinia virus, an orthopoxvirus, is the most widely studied of the poxviruses, and is the subject of U.S. Pat. No. 4,603,112 (Paoletti et al.,). Fowlpox virus is an avipoxvirus or avian poxvirus.
Recent advances in recombinant DNA technology have allowed vaccinia virus to be used as a vector to carry and express foreign genes. For a review see M. Mackett & G. L. Smith, Journal of General Virology 67, 2067-2082 (1986). Certain properties of vaccinia virus make it suitable for this purpose. Firstly, it tolerates large amounts of extra DNA in its genome, at least up to 25,000 base pairs. Secondly, it encodes its own RNA polymerase which specifically initiates transcription of messenger RNA, beginning at the viral promoter sequences on the DNA genome. The host cell RNA polymerase II does not recognise these viral promoters, nor does the vaccinia RAN polymerase transcribe from promoters recognised by the host cell RNA polymerase. These two properties allow foreign genes to be inserted into the vaccinia virus genome under the control of a vaccinia virus promoter. Because of the very large size of the vaccinia virus genome (186,000 base pairs) and the fact that the DNA alone is not infectious, conventional recombinant DNA techniques of restriction enzyme cleavage and ligation of DNA fragments into the genome are not technically feasible. Therefore DNA is introduced into the genome by a process of homologous recombination. Homologous recombination involves essentially (1) pre-selecting a length of the vaccinia virus (VV) genome in some region which does not impair the replication and normal functioning of the virus (hereinafter called a "non-essential region"), (2) making a construct of a length of foreign DNA in a copy of the non-essential region so that the foreign DNA is flanked by extensive sequences of non-essential region of VV DNA, (3) co-infecting appropriate tissue culture cells with the VV and the construct and (4) selecting cells containing VV in which the pre-selected length has been swapped over ("recombined") in vivo so that it is replaced in the genome by the construct DNA.
In order to insert the foreign gene in to the construct, the construct should itself be contained in a vector, e.g. a plasmid. It should also comprise a promoter for regulating expression of the foreign DNA within the virus. The procedure is more fully described in the Mackett and Smith review supra. Vaccinia virus vectors have been used in this way experimentally for the expression of DNA for several viral proteins. See, for example, M. Kieny et al., Nature 312, 163-166 (1984) on the expression of a rabies virus glycoprotein. Since the vaccinia virus vector can be attenuated, i.e. altered to make it less virulent, without impairing its use as a vector, it has considerable potential for use in vaccination.
It has been recognised for some years that in principle similar technology could be applied to fowlpox virus (FPV), see, for example, M. M. Binns et al., Israel Journal of Veterinary Medicine 42, 124-127 (1986), thereby providing a vector for use in vaccinating poultry. FPV like VV, has a genome of vast size (it is even larger than VV: estimates range from 240 to 360 kilobases) and it is not known to what extent it is similar to vaccinia virus.
One of the essential requirements for the expression of foreign DNA in a FPV vector is a strong promoter, which will be recognised by the FPV RNA polymerase. Several promoters have been identified in VV but their relative strengths have not been fully explored. The main ones are as follows:
1. p7.5. The 7.5 Kd polypeptide promoter, which has early and late activities, has been widely used to express genes inserted into vaccinia, S. Venkatesan et al., Cell 125, 805-813 (1981), M. A. Cochran et al., J. Virol. 54, 30-37 (1985). PA0 2. p11. The gene for the 11 Kd major structural polypeptide, mapping at junction of vaccinia HindIII fragments F/E, has late promoter which has been widely used, C. Bertholet et al. Proc. Natl. Acad. Sci. USA 82, 2096-2100, (1985). PA0 3. pTK. Promotes the thymidine kinase, gene which maps in vaccinia HindIII fragment J, J. P. Weir et al., Virology 158 206-210 (1987). This promoter has not been used much and is thought not to be strong. PA0 4. pF. Promotes an unknown, early, non-essential gene, which maps in vaccinia HindIII fragment F, see D. Panicali et al. Proc. Natl. Acad. Sci. USA 80, 5364-5368 (1983). It has recently shown to be "relatively inefficient" i.e. 10-fold lower than the TK promoter, B. E. H. Coupar et al., J. Gen. Virol. 68, 2299-2309 (1987). PA0 5. p4b. The 4b gene encodes a 62 Kd core protein. It has a late promoter which maps in vaccinia HindIII fragment A, see J. Rosel et al., J. Virol. 56, 830-838 (1985). The 4b protein accounts for approx 10% of viral protein in vaccinia. PA0 6 and 7. pM. and pI. These are two uncharactised early vaccinia promoters from vaccinia HindIII M and I fragments respectively used in construction of a multivalent vaccinia vaccine, M. E. Perkus et al., Science 229, 981-984 (1985). PA0 8. p28K. Promotes a gene encoding a later 28 Kd core protein, J. P. Weir et al., J. Virol. 61, 75-80 (1987). It hasn't been used much. PA0 (1) The FP4b gene which encodes a protein of about 657 amino acids in a sequence beginning ##STR5## (2) The BamHI fragment ORF8 gene encoding a protein of about 116 amino acids in a sequence beginning ##STR6## (3) The BamHI fragment ORF5 gene encoding a protein of about 105 amino acids in a sequence beginning ##STR7## (4) The BamHI fragment ORF10 gene encoding a protein of about 280 amino acids in a sequence beginning ##STR8## PA0 (1) a first homologously recombinable sequence of the fowlpox virus (FPV) genome, PA0 (2) a sequence within a first portion of a non-essential region (NER) of the FPV genome, PA0 (3) FPV promoter DNA according to the invention, PA0 (4) a foreign gene transcribably downstream of the promoter (whereby when the fowlpox virus RNA polymerase binds to the promoter it will transcribe the foreign gene into mRNA) and PA0 (5) a sequence within a second portion of the same NER of the FPV genome, the first and second sequences preferably being in the same relative orientation as are the first and second portions of the NER within the FPV genome, and PA0 (6) a second homologously recombinable sequence of the FPV genome, said sequences (1) and (6) flanking the NER in the FPV genome and being in the same relative orientation in the recombination vector as they are within the FPV genome.
Because of the lack of information about the genomic DNA sequence of FPV (and, indeed, VV, since only about a third of the genomic DNA sequence of VV has been published), it has not been possible to predict whether a particular promoter known in VV has a counterpart in FPV, nor could its efficiency as a promoter be predicted.
Only very limited data have been published about the DNA sequence of the FPV genome. Thus, D. B. Boyle et al., Virology 156, 355-365 (1987), have published the sequence of the thymidine kinase (TK) gene and flanking sequence totalling 1061 base pairs. These authors looked at the FPV TK promoter region and noted that it contained a so-called consensus sequence common to eleven VV gene promoters [A. Plucienniczak et al., Nucleic Acids Research 13, 985-988 (1985)]. This "consensus sequence" is supposedly based on TATA--(20 to 24 bp)--AATAA, but there were many divergences from it and the whole region is so AT-rich that the notion of a "consensus sequence" does not bear critical examination. Moreover, the distances between these consensus sequences and the 5' ends of the TK mRNAS differed as between FPV and VV. Since the FPV TK gene was found to be expressed in vaccinia virus vector, and therefore recognised by the VV RNA polymerase, some degree of similarity between these two promoters is deducible. It does not follow, of course, that every VV promoter would be highly homologous with every FPV promoter and indeed unpublished data of the present inventors suggests that this is not the case.
Further prior art is referred to below after the section "Summary of the Invention", without which its context would not be apparent.